Antigen composition against mycoplasma

ABSTRACT

The present invention relates to a putative protective antigen against a Mycoplasma, prepared by a method including a sample of a Mycoplasma; an antibody probe including at least one antibody against a Mycoplasma produced by a method including: providing a biological sample taken a short time after an immune animal has been challenged with a Mycoplasma or Mycoplasma extract taken from the infection site or an area of a lesion or an area close to the infection site or lesion; isolating cells from the biological sample; culturing cells in vitro in a suitable culture medium; and harvesting antibodies produced from said cells; probing the Mycoplasma sample with the antibody probe to detect at least one antigen; and isolating the antigen detected, also including diagnostic antigens, the preparation thereof, and their use in the formation of vaccine compositions, particularly vaccine compositions against  Mycoplasma hyopneumoniae  infections.

[0001] The present invention relates to protective and diagnosticantigens, the preparation thereof, and their use in the formation ofvaccine compositions, particularly vaccine compositions againstMycoplasma hyopneumoniae infections.

[0002]Mycoplasma hyopneumoniae is a ubiquitous swine respiratorypathogen causing mycoplasmal pneumoniae in swine (swine enzooticpneumonia). Swine enzootic pneumonia is probably the most widespread andeconomically significant disease in swine producing countries of theworld. The economic effects of swine enzootic pneumonia (SEP) arecomplex, and the cost of the disease is severe. In Australia, thedisease was estimated in 1988 to cost approximately $20,000,000 perannum. Increased mortality, decreased growth weight, depressed feedconversion, susceptibility to secondary bacterial infections, increasedmanagement costs, and increased use of antibiotics, are the main reasonsfor the economic impact of SEP.

[0003] Whilst several experimental vaccines have been produced, thesehave resulted in less than optimal results, and utilising variousclasses of antibiotics such as tetracycline, lincamycin and tiamulin isstill the most widespread control treatment. Such antibiotics are,however of limited therapeutic value, because they do not prevent theestablishment of an infection and lung lesions may develop aftertreatment ends.

[0004] European Patent Application 359 919 to ML Technology VenturesL.P. describes a series of antigens, 36 kD, 41 kD 74.6 kD and 96 kD insize, and proposes the use of such antigens in vaccines. Resultspresented suggest that some protection in pigs against challenge wasachieved.

[0005] However, there remains a need in the art for an effective vaccineagainst M. hyopneumoniae which would confer protection againstcolonisation and clinical disease following M. hyopneumoniae challengeand also significantly reduce the morbidity and mortality from secondaryinfections.

[0006] Accordingly, it is an object of the present invention toovercome, or at least alleviate, one or more of the difficulties anddeficiencies in the prior art.

[0007] Accordingly, in a first aspect of the present invention there isprovided a putative protective antigen against a Mycoplasma, preferablyMycoplasma hyopneumoniae prepared by a method including

[0008] providing

[0009] a sample of a Mycoplasma;

[0010] an antibody probe including at least one antibody against aMycoplasma produced by a method including:

[0011] providing a biological sample taken a short time after an immuneanimal has been challenged with a Mycoplasma or Mycoplasma extract takenfrom the infection site or an area of a lesion or an area close to theinfection site or lesion;

[0012] isolating cells from the biological sample;

[0013] culturing cells in vitro in a suitable culture medium; and

[0014] harvesting antibodies produced from said cells;

[0015] probing the Mycoplasma sample with the antibody probe to detectat least one antigen; and

[0016] isolating the antigen detected.

[0017] The protective antigens may also function as diagnostic antigensas discussed below.

[0018] Accordingly, in a preferred aspect of the present invention thereis provided a putative protective antigen against Mycoplasmahyopneumoniae, or related infections, selected from the group ofantigens having approximate molecular weights of 110-114, 90-94 72-75,60-64, 52-54 and 46-48 kilodaltons (kD), as hereinafter described,mutants, derivatives and fragments thereof. The putative protectiveantigen may be a surface protein. The putative protective antigen may bea surface lipoprotein or membrane protein.

[0019] Preferably the protective antigens are selected from the group ofantigens having approximate molecular weights of 110-114, 90-94, 74, 62,52 and 48 kD.

[0020] Preferably, the 72-75 kD antigen includes the followingN-terminal amino acid sequence:

[0021] AGXLQKNSLLEEVWYLAL

[0022] and, optionally, one or more of the following internal amino acidsequences:

[0023] AKNFDFAPSIQGYKKIAHEL

[0024] NLKPEQILQLLG

[0025] LLKAEXNKXIEEINTXLDN

[0026] Preferably, the 60-64 kD antigen includes one of the followingN-terminal amino acid sequences:

[0027] MKLAKLLKGFX(N/L)(M/V)IK

[0028] ADP(F/I)(R/E)Y(V/A)PQG(Q/A)X(M/N)VG

[0029] Preferably, the 52-54 kD antigen includes the followingN-terminal amino acid sequence:

[0030] AGXWAKETTKEEKS

[0031] and, optionally, one or more of the following internal amino acidsequences:

[0032] AWVTADGTVN

[0033] AIVTADGTVNDNKPNQWVRKY.

[0034] Preferably, the 46-48 kD antigen includes the followingN-terminal amino acid sequence:

[0035] AGXGQTESGSTSDSKPQAETLKHKV

[0036] and, optionally, one or more of the following internal amino acidsequences:

[0037] TIYKPDKVLGKVAVEVLRVLIAKKNKASR

[0038] AEQAITKLKLEGFDTQ

[0039] KNSQNKIIDLSPEG

[0040] The 46-48 kD antigen may be encoded by a nucleic acid fragment:10                 20         30         40         50 12345678901234567890 1234567890 1234567890 1234567890 ATGAAAAAAA TGACACTATACCAGAGGAAA GAGCAGTATA TAAAATAATT 50 AAAATTACAT TTTCTTCAAT TGCGCCAGAATTTTTAAGAA TTAGTACATT 100 AAAAAGTAGA ACAAAAGTTA TTAATGTAAA CATTAGCGCAATCCTTAAGA 150 AAAAATTAAA AGTTTTATCT ATTTTTTTTA ATCGAAATCC AACCAGGCAT200 AAATCTTTGT CAGTATTTAT CAAGTCGGTA TTTTTTCATT ATTTCTACTA 250AAATATTATT TGAATTTGCA TTTTCCATAA TCTAAAATTT TACATTTTTT 200 TATAACAATTTTTAAAAATT ACTCTTTAAT TTATAGTATT TTTTTATTTT 350 TTAGTCTAAA TTATAAAATTATCTTGAATT TTATTTGAAT TTTTATAATT 400 TAGTACTAAA AAATACAAAT ATTTTTTACTATTCTAAGAA AAATTCATTT 450 TTAAAAAAAA ATTGATTTTT ATAGTATAAT TTGTTTGTATAATTGAATTA 500 ACTTGATTTG AAAGGGAACA AAATGAAAAA AATGCTTAGA AAAAAATTCT550 TGTATTCATC AGCTATTTAT GCAACTTCGC TTGCATCAAT TATTGCATTT 500GTTGCAGCAG GTTGTGGACA GACAGAATCA GGTTCAACTT CTGATTCTAA 550 ACCACAAGCGGAGACGCTAA AACATAAAGT AAGTAATGAT TCTATTCGAA 700 TAGCACTAAC CGATCCGGATAATCCTCGAT GAATTAGTGC CCAAAAAGAT 750 ATTATTTCTT ATGTTGATGA AACAGAGGCAACAACTTCAA CAATTACAAA 800 AAACCAGGAT GCACAAAATA AATGACTCAC TCAGCAAGCTAATTTAAGCC 850 CAGCGCCAAA AGGATTTATT ATTGCCCCTG AAAATGGAAC TGGAGTTGGA900 ACTGCTGTTA ATACAATTGC TGATAAACGA ATTCCGATTG TTGCCTATGA 950TCGACTAATT ACTGGATCTG ATAAATATGA TTGGTATGTT TCTTTTGATA 1000 ATGAAAAAGTTGGTGAATTA CAAGGTCTTT CACTTGCTGC GGATCTATTA 1050 GGAAAAGAAG ATGGTGCTTTTGATTCAATT GATCAAATGA ATGAATATCT 1100 AAAATCACAT ATGCCCCAAG AGACAATTTCTTTTTATACA ATCGCGGGTT 1150 CCCAAGATGA TAATAATTCC CAATATTTTT ATAATGGTGCAATGAAAGTA 1200 CTTAAAGAAT TAATGAAAAA TTCGCAAAAT AAAATAATTG ATTAATCTCC1250 TGAAGGCGAA AATGCTGTTT ATGTCCCAGG ATGAAATTAT GGAACTGCCG 1300GTCAAAGAAT CCAATCTTTT CTAACAATTA ACAAAGATCC AGCAGGTGGT 1350 AATAAAATCAAAGCTGTTGG TTCAAACCAG GCTTCTATTT TCAAAGGATT 1400 TCTTGCCCCA AATGATGGAATGGCCGAACA AGCAATCACC AAATTAAAAC 1450 TTGAAGGGTT TGATACCCAA AAAATCTTTCTAACTCGTCA AGATTATAAT 1500 GATAAAGCCA AAACTTTTAT CAAAGACGGC GATCAAAATATGACAATTTA 1550 TAAACCTGAT AAAGTTTTAG GAAAAGTTGC TGTTGAAGTT CTTCGGGTTT1600 TAATTGCAAA GAAAAATAAA GCATCTAGAT CAGAAGTCGA AAACGAACTA 1650AAAGCAAAAC TACCAAATAT TTCATTTAAA TATGATAATC AAACATATAA 1700 AGTACAAGGTAAAAATATTA ATACAATTTT AGTAAGTCCA GTAATTGTTA 1750 CAAAAGCTAA TGTTGATAATCCTGATGCCT AA 1732

[0041] Accordingly, in a further aspect the present invention providesan isolated nucleic acid fragment encoding a putative protective antigenagainst Mycoplasma hyopneumoniae or related infections, said nucleicacid fragment: 10                 20         30         40         501234567890 1234567890 1234567890 1234567890 1234567890 ATGAAAAAAATGCCACTATA CCAGAGGAAA GAGCAATATA TAAAATAATT 50 AAAATTACAT TTTCTTCATTTGCGCCAGAA TTTTTAAGAA TTAGTACATT 100 AAAAAGTAGA ACAAAAGTTA TTAATGTAAACATTAGCACA ATCCTTAAGA 150 AAAAATTAAA AGTTTTATCT ATTTTTTTTA TCGAAAATCCAACCAGGCAT 200 AAATCTTTGT CAGTATTTAT CAAGTCGGTA TTTTTTCATT ATTTCTACTA250 AAATATTATT TGAATTTGCA TTTTCCATAA TCTAAAAATT TACATTTTTT 300TATAACAATT TTTAAAAATT ACTCTTTAAT TTATAGTATT TTTTTATTTT 350 TTAGTCTAAATTATAAAATT ATCTTGAATT TTATTTGAAT TTTTATAATT 400 TAGTACTAAA AAATACAAATATTTTTTCCT ATTCTAAGAA AAATTCAATT 450 TTTAAAAAAA ATTGATTTTT ATAGTATAATTTGTTTGTAT AATTGAATTA 500 ACTTGATTTG AAAGGGAACA AATAAAAAAA AATGCTTAGAAAAAAATTCT 550 TGTATTCATC AGCTATTTAT GCAACTTCGC TTGCATCAAT TATTGCATTT500 GTTGCAGCAG GTTGTGGACA GACAAAATCA GGTTCAACTT CTGATTCTAA 550ACCACAAGCC GAGACGCTAA AACATAAAGT AAGTAATGAT TCTATTCGAA 700 TAGCACTAACCGATCCAGAT AATCCTCGAT GAATTAGTGC CCAAAAAGAT 750 ATTATTTCTT ATGTTAATAAAACAAAGGCA GCAACTTCAA CAATTACAAA 800 AAACCAGAAT GCACAAAATA ACTGACTCACTCAGCAAGCT AATTTAAGCC 850 CAGCGCCAAA AGGATTTATT ATTGCCCCTG AAAATGGAAGTGGAGTTGGA 900 ACTCCTGTTA ATACAATTGC TGATAAAGGA ATTCCGATTG TTGCCTATGA950 TCGACTAATT ACTGGATCTG ATAAATATAA TTGGTATGTT TCTTTTGATA 1000ATAGAAAAGT TGGTGAATTA CAAAGTCTTT CACTTGCTGC GGGTCTATTA 1050 GGAAAAGAAGATGGTGCTTT TGATTCAATT GATCAAATGA ATGAATATCT 1100 AAAATCACAT ATGCCCCAAGAGACAATTTC TTTTTATACA ATCGCGGGTT 1150 CCCAAGATGA TAATAATTCC CAATATTTTTATAATGGTGC AATGAAAGTA 1200 CTTAAAGAAT TAATGAAAAA TTCGCAAAAT AAAATAATTGATTTATCTCC 1250 TGAAGGCGAA AATGCTGTTT ATGTCCCAGG ATGAAATTAT GGAACTGCCG1300 GTCAAAGAAT CCAATCTTTT CTAACAATTA ACAAAGATCC AGCAGGTGGT 1350AATAAAATCA AAGCTGTTGG TTCAAAACCA GCTTCTATTT TCAAAGGATT 1400 TCTTGCCCCAAATGATCGAA TGGCCAAACA AGCAATCACC AAATTAAAAC 1450 TTGAAGGGTT TGATACCCAAAAAATCTTTG TAACTCGTCA ACATTATAAT 1500 GATAAAGCCA AAACTTTTAT CAAAGACGGCGATCAAAATA TGACAATTTA 1550 TAAACCTGAT AAAGTTTTAG GAAAAGTTGC TGTTGAAGTTCTTCGGGTAT 1600 TAATTGCAAA GAAAAATAAA GCATCTAGAT CAGAAGTCGA TAACGAACTA1650 AAAGCAAAAC TACCAAATAT TTCATTTAAA TATGATAATC AAACATATAA 1700AGTACAAGGT AAAAATATTA ATACAATTTT AGTAAGTCAA GTAATTGTCA 1750 CAAAAGCTAATGTTGATAAT CCTGATGCCT AA 1752

[0042] As cross protection between various Mycoplasma such as M.hyorhinis and M. synoviae has been documented, similar antigens may alsobe detected in other Mycoplasma species (FIG. 1).

[0043] In a still further aspect the present invention provides a methodfor preventing Mycoplasma infection in animals. Preferably theMycoplasma disease is a Mycoplasma hyopneumoniae disease such as swineenzootic pneumonia (SEP). This method includes administering to ananimal an effective amount of at least one protective antigen againstMycoplasma as described above.

[0044] The present invention further provides a vaccine compositionincluding a prophylactically effective amount of at least one putativeprotective antigen against a Mycoplasma as herein described. Preferablythe veterinary composition includes two or more putative protectiveantigens as herein described.

[0045] Accordingly in a preferred aspect the present invention providesa vaccine composition including two or more putative protective antigensselected from antigens having approximate molecular weights of 110-114,90-94, 72-75, 60-64, 52-54 and 46-48 kilodaltons.

[0046] The vaccine composition may include any combination of two ormore putative protective antigens selected from antigens havingapproximate molecular weights of 110-114, 90-94, 72-75, 60-64, 52-54 and46-48 kD. The two or more antigens may be selected from antigens fallingwithin one of the specified approximate molecular weights and/orantigens from different specified approximate molecular weights. Thecomposition may contain 3, 4, 5 or 6 antigens selected from protectiveantigens having molecular weights of approximately 110-114, 90-94,72-75, 60-64, 52-54 and 46-48 kD.

[0047] The vaccine compositions according to the present invention maybe administered orally or may be administered parenterally (for exampleby intramuscular, subcutaneous, intradermal or intravenous injection).The amount required will vary with the antigenicity of the activeingredient and need only be an amount sufficient to induce an immuneresponse typical of existing vaccines.

[0048] Reactive experimentation will easily establish the requiredamount. Typical initial doses of vaccine or veterinary compositions maybe approximately 0.001-1 mg active ingredient/kg body weight. The doserate may increase or multiple doses may be used as needed to provide thedesired level of protection.

[0049] The vaccine composition according to the present invention mayfurther include a veterinary acceptable carrier, diluent or excipienttherefor. Preferably the active ingredient may be suspended or dissolvedin a carrier. The carrier may be any solid or solvent that is nontoxicto the animal and compatible with the active ingredient. Suitablecarriers include liquid carriers, such as normal saline and othernontoxic salts at or near phsyiological concentrations, and solidcarriers, such as talc or sucrose.

[0050] Preferably the vaccine contains an adjuvant, such as Freund'sadjuvant, complete or incomplete, or immunomodulators such as cytokinesmay be added to enhance the antigenicity of the antigen if desired.

[0051] More preferably the adjuvant is of the mineral-oil type as thesehave been found to be consistently superior at inducing antibody titresand Delayed Type Hypersensitivity responses. A particularly preferredadjuvant is that marketed under the trade designation Montanide ISA-50and available from Seppic, Paris, France.

[0052] When used for administering via the bronchial tubes, the vaccineis suitably present in the form of an aerosol.

[0053] In a still further aspect of the present invention there isprovided a diagnostic kit including a diagnostic antigen against aMycoplasma, preferably Mycoplasma hyopneumoniae, identified and purifiedas described above.

[0054] The putative protective antigens according to the presentinvention may be isolated and identified utilising the general methodsdescribed in Australian patent application 49035/90, the entiredisclosure of which is incorporated herein by reference.

[0055] Accordingly, in a further aspect, the present invention providesa method for producing at least one antibody against a Mycoplasma. Thismethod includes

[0056] providing a biological sample taken a short time after an immuneanimal has been challenged with a Mycoplasma or Mycoplasma extract takenfrom the infection site or an area of a lesion or an area close to theinfection site or lesion;

[0057] isolating cells from the biological sample;

[0058] culturing cells in vitro in a suitable culture medium; and

[0059] harvesting antibodies produced from said cells.

[0060] The Mycoplasma may be Mycoplasma hyopneumoniae.

[0061] The animal may be a mammal including humans. The mammal may be adomestic animal such as a pig, sheep or cattle.

[0062] The biological animal sample may be of any suitable type. Thebiological sample may be taken from animal tissue, organs, lymph orlymph nodes. The biological sample may be taken from the infection site,the lungs of the animal, or an area of a lesion which may be formed oran area close to the infected site or a lesion such as in the lymphnodes draining from the lungs.

[0063] However, serum/plasma samples are not used as the biologicalsamples according to this aspect of the present invention. It has beenfound that the majority of antibodies found in a serum/plasma sample areirrelevant to protection or specific diagnosis or a Mycoplasma or areunrelated to the Mycoplasma. In addition, other serum/plasma componentsmay interfere with the specific reactions between pathogen componentsand antibodies to them.

[0064] In contrast, the probes described in the present invention arehighly enriched in Mycoplasma-specific antibodies of particularimportance to protective immunity.

[0065] It is preferred that the biological samples are taken from theanimals at a predetermined time in the development of the disease. Ingeneral, for a Mycoplasma infection, it has been found that thebiological samples should be taken approximately 2 to 7 days afterchallenge with or after administration of products obtained from apathogen or with the pathogen itself.

[0066] The cells isolated from the biological sample may include Bcells.

[0067] Thus, preferably the cells are taken a short time after in vivostimulation, preferably within approximately 2 to 5 days thereafter,resulting in the in vivo induction of antibody forming cells which willsecrete specific antibodies into the culture medium after in vitroincubation.

[0068] In vitro secretion of antibodies in the culture medium byrecently activated B cells may be enhanced by the addition of helperfactors to the cultures. The helper factors may be cytokines used aloneor in combination, including Interleukin 1, 2, 3, 4, 5, 6, 7 and 8,colony stimulating factors, interferons and any other factors that maybe shown to have an enhancing effect on specific B cell secretion.

[0069] The method of producing an antibody may include a further step ofactivating the cells isolated to proliferate and secrete and/or releaseantibodies.

[0070] The cell activation step may include adding a cell activatingagent to the culture medium. The cell activating agent may be selectedfrom mitogens and helper factors produced by leukocytes, or theirsynthetic equivalents or combinations thereof.

[0071] The mitogens may be selected from the group including productsderived from pokeweed (Phytolacca americana) also known as pokeweedmitogen (PWM), polyvinylpyrrolidone (PVP), polyadenylic-polyuridylicacid (poly(A-U)), purified protein derivate (PPD),polyinosinic-polycytidilic acid (poly(I-C)), lipopolysaccharide (LPS),staphylococcal organisms or products thereof. Bacto-streptolysin Oreagent (SLO), Staphylococcal phage lysate (SPL), Epstein-Barr virus(EBV), Nocardia water-soluble mitogen (NWEM), phytohemagglutinin (PHA),Concanavalin A (Con A), and dextran-sulphate and mixtures thereof. Thecell proliferation agent may be any agent that indirectly or directlyresults in B cell proliferation and/or antibody secretion such assolid-phase anti-immunoglobulin. The helper factors may be selected fromthe group including cytokines including interleukin 1, 2, 3, 4, 5, 6, 7and 8, colony stimulating factors, interferons and any other helperfactors that may be shown when added alone, or in combination with otherfactors and agents, to have an enhancing effect on specific B cellproliferation and/or antibody secretion. This in no way is meant to bean exhaustive list of mitogens and cell actuating agents includinghelper factors.

[0072] The in vitro culturing of the cells may be conducted with orwithout prior steps to separate sub-populations of cells. The harvestingof antibodies may be conducted by harvesting of the supernatant from theculture medium. This supernatant contains antibodies secreted by thesecells during the in vitro culture or artificially released from the Bcells, for example by lysis of the B cells. It has been found that theantibody-containing supernatants may be used directly to detect antigensof the Mycoplasma.

[0073] In a preferred aspect of the present invention, there is provideda method for identifying an antigen associated with a Mycoplasma,preferably Mycoplasma hyopneumoniae. This method includes

[0074] providing

[0075] a sample of a Mycoplasma; and

[0076] an antibody probe including at least one antibody against aMycoplasma;

[0077] probing the Mycoplasma sample with the antibody probe to detectat least one antigen; and

[0078] isolating the antigen detected.

[0079] The sample of Mycoplasma may be mixed with a standard buffersolution and placed on a standard support such as an SDS-polyacrylamidegel to separate the proteins contained thereon (FIG. 2).

[0080] Alternatively the proteins may be selected utilising thenon-ionic detergent Triton X-114 (TX-114). Insoluble material may beremoved by centrifugation. Proteins soluble in the TX-114 phase may thenbe precipitated out (FIG. 2).

[0081] The separate proteins may then be transferred to nitrocellulose,nylon or other sheets.

[0082] The probing with a suitable antibody may further includesubjecting the product produced thereby to a detection assay. Thedetection assay may include Western blot techniques. The detection assaymay be an immunoprecipitation assay a radioimmunoassay, an enzyme-linkedimmunoassay or immunofluorescent assay (FIGS. 3, 4 and 5).

[0083] The antibody produced as described above may be utilized simplyin the form of the supernatant harvested from the culture medium.Alternatively, the antibodies may be separated and purified.

[0084] In a further preferred aspect of the present invention theantibody contained in the culture medium may be used for the affinitypurification, preferably immuno-affinity purification of antigen.

[0085] Accordingly, in a preferred aspect there is provided a method forpurifying antigen. This method includes

[0086] providing

[0087] a crude antigen mixture; and

[0088] an antibody against a Mycoplasma immobilized on a suitablesupport;

[0089] subjecting the crude antigen mixture to affinity chromatographyutilizing the immobilized antibody; and

[0090] isolating the purified antigen so formed.

[0091] The antibody is produced by the method described above.

[0092] Antibody can be obtained from the culture supernatant probe byconventional methods. For example, methods usually used to purifyimmunoglobulins from serum or plasma, e.g. precipitation with ammoniumsulphate, fractionation with caprylic acid, ion exchange chromatography,or by binding and elution from immobilized protein G or protein A, maybe utilized. Antibody so obtained can then be coupled to suitablesupports. e.g., CNBr-activated Sepharose 4S (Pharmacia), Affi-gel(Bio-RAD), or other affinity chromatography supports able to bindproteins.

[0093] Immobilized antibody can then be applied to the fractionation andpurification of specific antigen from a complex Mycoplasma extract byaffinity chromatography. After binding of antigen to immobilizedantibody, unbound macromolecular species can be washed away from thesolid support with, e.g. buffers containing 1.5 M NaCl. Subsequently theantigen can be eluted from the affinity column with, e.g. low or high pHbuffer or buffers containing chaotropic ions, e.g. 0.5-3.0 M sodiumthiocyanate.

[0094] The application of the antibody probe to affinity chromatographyenables sufficient quantities of specific antigens to be rapidlyisolated from a complex crude extraction mixture for biochemicalcharacterization, amino-acid sequencing and vaccination of animal forlimited protection studies. Application of affinity chromatography forobtaining antigen(s) avoids the difficulties often encountered whenapplying conventional biochemical techniques to the purification of anantigen about which little or no data is known. It also obviates theneed to raise polyclonal or monoclonal antibodies for the purpose of“analytical” affinity chromatography. Large scale preparation may,however, require the preparation of polyclonal or monoclonal antibodies.

[0095] Having identified the antigen(s) molecular biology, chemicaltechniques, e.g. cloning techniques, may be used to produce unlimitedamounts of this antigen or, alternatively, synthetic peptidescorresponding to different fragments of the identified antigens may beused as a means to produce a vaccine.

[0096] Accordingly in a preferred aspect of the present invention thereis provided a method for preparing a synthetic antigenic polypeptideagainst Mycoplasma, preferably Mycoplasma hyopneumoniae, which methodincludes

[0097] providing

[0098] a cDNA library or genomic library derived from a sample ofMycoplasma; and

[0099] an antibody probe as described above;

[0100] generating synthetic polypeptides from the cDNA library orgenomic library;

[0101] probing the synthetic polypeptides with the antibody probe; and

[0102] isolating the synthetic antigenic polypeptide detected thereby.

[0103] Either cDNA or genomic libraries may be used. The cDNA or genomiclibraries may be assembled into suitable expression vectors that willenable transcription and the subsequent expression of the clone cDNA,either in prokaryotic hosts (e.g. bacteria) or eukaryotic hosts (e.g.mammalian cells). The probes may preferably be selected from

[0104] (i) synthetic oligonucleotide probes based on the amino acidsequence of the antigen identified and purified as described above;

[0105] (ii) antibodies obtained from the culture medium produced asdescribed above;

[0106] (iii) monoclonal or polyclonal antibodies produced against theantigens identified and purified as described above;

[0107] (iv) recombinant or synthetic monoclonal antibodies orpolypeptides with specificity for the antigen, e.g. as described by Wardet al., Nature, 241, pages 544-546 (1989).

[0108] The synthetic antigenic polypeptide produced in accordance withthe invention may be a fusion protein containing the synthetic antigenicpeptide and another protein.

[0109] In a further aspect of the present invention there is provided aDNA fragment encoding a putative protective antigen against Mycoplasmaor related infections, said DNA fragments having a nucleic acid sequenceaccording to FIGS. 6a and 6 b or an homologous sequence and functionallyactive fragments thereof.

[0110] In a further preferred aspect of the present invention there isprovided a clone including a DNA fragment encoding a putative protectiveantigen against Mycoplasma or related infections, said DNA fragmentshaving a nucleic acid sequence according to FIGS. 6a and 6 b or anhomologous sequence and functionally active fragments thereof.

[0111] Preferably the clone is pC1-2.

[0112] The present invention will now be more fully described withreference to the accompanying Examples and drawings. It should beunderstood, however, that the description following is illustrative onlyand should not be taken in any way as a restriction on the generality ofthe invention described above.

[0113] In the figures:

[0114]FIG. 1: SDS-Polyacrylamide gel (12.5%) profiles of SDS extracts ofspecies of mycoplasma—Coomassie R250 stained. Lane 1 Pre-stainedMolecular Weight Standards Lane 2 M. gellisepticum Lane 3 M. synoviae.Lane 4 M. hyopneumoniae. Lane 5 M. hyorhinis. Lane 6 M. flocculare.

[0115]FIG. 2: SDS-Polyacrylamide gel (12.5%) profiles of extracts ofstrains of M. hyopneumoniae—Coomassie R250 stained gel Lane 1Pre-stained Molecular Weight Standards. Lane 2 Triton X-114 extract ofM. hyopneumoniae - strain Beaufort. Lane 3 As for Lane 2. Lane 4 SDSextract of M. hyopneumoniae strain Beaufort. Lane 5 SDS extract of M.hyopneumoniae strain 10110.

[0116]FIG. 3: Western blots of Triton X-114 extracted antigens from M.hyopneumoniae strain Beaufort, probed with serum and supernatantantibody probes. Lane 1 No antibody control Lane 2 Dookie pig serumcontrol 1/200. Lane 3 Pig 105 supernatant. Lane 4 Pig 1 supernatant.Lane 5 Dookie pig supernatant.

[0117]FIG. 4: Western blots of SDS extracted antigens from M.hyopneumoniae strain Beaufort probed with paired serum andsupernatantantibody probes. Fractionation of antigens on SDSPolyacrylamide gel (12.5%). Lane 1 a) Pig 453 supernatant. b) Pig 453serum 1/100. Lane 2 a) Pig 105 supernatant. b) Pig 105 serum 1/100. Lane3 a) Pig 1 supernatant. b) Pig 1 serum1/100. Lane 4 a) Pig 15supernatant. b) Pig 15 serum 1/100. Lane 5 a) Dookie supernatant. b)Dookie serum 1/100. Lane 6 No antibody control.

[0118]FIG. 5: Western blots of SDS extracted antigens from M.hyopneumoniae strain Beaufort probed with paired serum andsupernatantantibody probes. Fractionation of antigens on SDSPolyacrylamide gel (10.0%). Lane 1 a) Pig 453 supernatant. b) Pig 453serum 1/100. Lane 2 a) Pig 105 supernatant. b) Pig 105 serum 1/100. Lane3 a) Pig 1 supernatant. b) Pig 1 serum 1/100. Lane 4 a) Pig 15supernatant. b) Pig 15 serum 1/100. Lane 5 a) Dookie supernatant. b)Dookie serum 1/100. Lane 6 No antibody control.

[0119]FIG. 6: The entire 48 k gene sequence.

[0120]FIG. 7: tHE 48 kDa protein sequence of the 48 k gene sequence.

EXAMPLE 1 Mycoplasma hyopneumoniae Media

[0121] Friss Media

[0122] Hovind-Hougen, K., Friss, N. F., Research in Veterinary Science,1991, 51, pp 156-163, “Morphological & Ultrastructural Studies of M.flocculare and M. hyopneumoniae in vitro”.

[0123] 250 ml Hanks BSS

[0124] 140 ml Water

[0125] 1.5 gm Brain Heart infusion

[0126] 1.6 gm PPLO Broth w/o CV

[0127] Autoclave at 120° C. for 20 minutes

[0128] 18 ml Yeast Extract (100 g YSC-2 Sigma in 750 ml)

[0129] 3.7 ml 0.2% DNA in 0.1% Na₂CL₃

[0130] 5.14 ml 1%-NAD

[0131] 0.6 ml 1% Phenol red

[0132] Adjust to pH 7.3 to 7.4

[0133] Filter through 0.45 um, 0.2 um membrane, store at 4° C.

[0134] Add sterile Horse or Pig serum to 20%

[0135] and Antibiotics prior to use

[0136] Etheridge Media

[0137] Etheridge, J. R., Cottew, G. S., Lloyd, L. C., AustralianVeterinary Journal, August 1979, 56, pp 356-359, “Isolation ofMycoplasma hyopneumoniae from lesions in experimentally infected pigs”.Materials For 600 mls Hanks BSS 18.9 ml Martleys Digest broth 1.28 gmHeart Infusion broth 1.65 gm Lactalbumin hydrolysate 2.21 gm Glucose4.41 gm Yeast Extract autolysate 8.82 ml Pig Serum (filtered) 163 ml 1%NAD 6.17 ml 1% Phenol red 1.32 ml 0.2% DNA in 0.1% Na₂CO₃ 4.41 ml

[0138] Make up to 600 ml with MQ water (about 350-400 ml)

[0139] Adjust pH to 7.4 and filter through: 3.0 um, 0.8 um 0.45 um, 0.2um.

[0140] Store at 4° C.

[0141] Development of Immune Sows

[0142] Cull sows and naive gilt (unmated sow designated Dookie).

[0143] Challenged on numerous occasions, with culture grown M.hyopneumoniae and lung homogenate. Given intranasally andintratracheally. Period of challenge—from September 1991 to Jan. 21,1992.

[0144] Tiamulin antibiotic given Jan. 31, 1992 to Feb. 4, 1992. Restedfor approximately 8 weeks.

[0145] Infectious Challenge

[0146] 120 ml of frozen culture of M. hyopneumoniae strain Beaufort spundown (12,000×g, 20 min.) and resuspended in 50 ml complete medium andcultured overnight at 37° C. The overnight culture was centrifuged(12,000×g, 20 min) and the Mycoplasma cells resuspended in 10 ml serumfree Mycoplasma culture medium. The 10 ml of concentrated mycoplasma wasadministered to anaesthetised immune sows via a catheter to ensure theinoculum was placed into the trachea.

[0147] Three of four days post-challenge, the sows were killed, andlymph nodes draining the lungs taken—these included the left and righttracheobronchial lymph nodes, and the lymph nodes located at thebifurcation of the trachea.

[0148] Antibody probes were prepared from pig lymph nodes and utilisedto detect putative protection antigens as described in Australian PatentApplication 49035/90 referred to above. Separate cell cultures wereobtained from individual lymph nodes. Culture supernatants wereharvested after 5 days of culture.

[0149] Antigen Preparation

[0150]Mycoplasma hyopneumoniae strain Beaufort was cultured in Etheridgemedia until the pH had dropped to between 6.8 and 7.0. Cells of M.hyopneumoniae were harvested from culture by centrifugation at 12,000×gfor 20 min., washed 4 times with either sterile PBS or 0.25 M NaCl andthen the pelleted cells extracted with one of the following.

[0151] (i) Sodium Dodecyl Sulphate (SDS)

[0152] The cell pellet was resuspended in 0.2% SDS and extracted for 2hours at 37° C. Insoluble material was pelleted from the extract at12,000×g for 10 min. and the soluble extract run on SDS-polyacrylamidegel electrophoresis (SDS-PAGE).

[0153] (ii) Triton X-114

[0154] The method of Bordier (J. Bio. Chem. 1981, 256:1604-1606) wasused to selectively extract membrane proteins using the non-ionicdetergent Triton X-114.

[0155] The cell pellet was resuspended in cold PBS to 2 mg/ml proteinand a cold pre-condensed solution of TX-114 added to give a finalconcentration of 1% (v/v) TX-114. Extraction was achieved by incubationovernight at 4° C. with gentle mixing. Insoluble material was removed bycentrifugation at 12,000×g for 20 min. at 4° C. The Triton X-114 solublemembrane proteins were then obtained by achieving a phase separation at37° C.

[0156] Proteins soluble in TX-114 phase were precipitated with 80%ethanol in the presence of carrier dextran (80,000 molecular weight) at−70° C. overnight. The proteins were collected by centrifugation at12,000×g for 30 min. and dissolved to 500 ug/ml in 4 M urea.

[0157] Identification of Antigens

[0158] Six antigens were identified utilising the above-mentionedtechnique. The identified antigens were those that were consistentlyidentified by the antibody probes from the immune cultures and theDookie gilt. The results are summarised in Table 1. TABLE 1 MolecularWeight (kD) Characteristics 110-114 SDS Extracted 90-94 SDS Extracted72-75 Triton X-114 Extracted  60-64** SDS Extracted. Partitions toaqueous phase of Triton X-114 extract. 52-54 Triton X-114 Extracted46-48 Triton X-114 Extracted

[0159] Molecular Weight (kD) Amino Acid Sequence 46-48 48 K N-Terminal:AGXGQTESGSTSDSKPQAETLKHKV 48 K CNBR F 1: TIYKPDKVLGKVAVEVLRVLIAKKNKASR48 K CNBR F 2: AEQAITKLKLEGFDTQ 48 K CNBR F 3: KNSQNKIIDLSPEG 52-54 52 KTerminal: AGXWAKETTKEEKS 52 K CNBR F 1: AWVTADGTVN 52 K CNBR F 2:AIVTADGTVNDNKPNQWVRKY 60-64 52 K N-Terminal: MKLAKLLKGFX (N/L)(M/V) IK60-64 52 K N-Terminal: ADP(F/I)(R/E)Y(V/A)PQG(Q/A)X(M/N)VG 72-75 74 KN-Terminal: AGXLQKNSLLEEVWYLAL 74 K CNBR F 1: AKNFDFAPSIQGYKKIAHEL 74 KCNBR F 2: NLKPEQILQLLG 74 K CNBR F 3: LLKAEXNKXIEEINTXLDN

[0160] PCR of 48 kDa Gene

[0161] Polymerase Chain Reaction (PCR) oligonucleotide primers weredesigned from the amino acid sequences obtained from the N-terminal andinternal cyanogen bromide (CNBr) derived peptides. Inosine (I) wassubstituted at positions of high redundancy. The following primers wereused in a standard PCR assay, run on a Bartelt Gene Machine Roboticthermal cycling instrument. Oligo 48 K CNBr F A: ACIAACGACGAGAAGCCICAGGC     T  T  A  A     A Oligo 48 K CNBr F 2: TTIAGCTTIGTGATIGCCTGCTC    AT    A     T  T           T Oligo 48 K CNBr F 3:AGGTCGATGATCTTCCAICC  AA  A  A  T  T      T  T

[0162] The resulting PCR products were visualised on a 1.5% agarose gel,excised, and purified using a Prep-a-Gene (BioRad). They were cloned bystandard techniques into a dideoxy tailed T-vector (Holton and Graham,Nucleic Acids Research 19, 1156, 1991) and the nucleic acid sequencedetermined. The PCR product, obtained from the reaction using primers F1and F2 shown above, was of approximately 810 base pairs and was shown bysequencing to code for the previously determined amino acid sequence ofthe purified native 46-48 kDa protein.

[0163] Genomic Clone Isolation at 48 k Gene

[0164] The entire 48 k gene and 48 kDa protein (FIGS. 6 and 7) has beenisolated and sequenced. The gene was obtained from an M. hyopneumoniaegenomic library made by digesting genomic DNA with the restrictionenzyme CLA 1 and ligating the fragments into the vector pBluescript(Stratagene). The ligated product was then electroporated intoEscherichia coli strain SURE (Stratagene) and the cells plated on LuriaBroth agar plates containing 100 μg/ml Ampicillin (LB-Amp). The librarywas screened by DNA hybridisation with a polymerase chain reaction (PCR)product specific for the 48 kDa protein. Positive clones were grown inLB-Amp, the cells harvested and the DNA isolated and partially sequencedfor confirmation.

[0165] The positive clone pC1-2 was entirely sequenced and the proteinsequence deduced. This was compared to the protein sequence obtainedfrom the N terminus and Cyanogen Bromide fragments of the 48 kDa proteinto show the that the gene encoded the desired protein.

[0166] Adjuvant Selection

[0167] Young piglets, 5-7 weeks of age, were immunised with identifiedantigen(s). The antigens include Triton X-114 extract and identifiedproteins of 46-48, 52-53, 60-64, 70-75, 90-94 and 110-114 kD, eithersingly or in combination. An immunising dose of antigen, containingbetween 6-100 μg protein, was given by intramuscular injection incombination with an adjuvant. An adjuvant is selected from

[0168] (i) Seppic Montanide ISA-50

[0169] (ii) Quill A and other derivatives of saponin,

[0170] (iii) oil in water emulsion employing a mineral oil such as BayolF/Anacel A,

[0171] (iv) oil in water emulsion employing a vegetable oil such as cornoil safflower oil or other with lecithin as emulsifier,

[0172] (v) aluminium hydroxide gel, and

[0173] (vi) nonionic block polymer such as Pluronic F-127 produced byBASF (U.S.A.).

[0174] Immunising doses were given at 2-4 week intervals, the number ofdoses being dependent on the adjuvant and amount of antigen, butpreferably 2 to 3 doses are given.

[0175] Adjuvants were treated on the basis of being able to induceantibody titres, as measured by ELISA, and by assessment of inducedcell-mediated immunity as tested by Delayed-Type Hypersensitivity (DTH)reaction.

[0176] The results clearly show that mineral-oil type adjuvants areconsistently superior at inducing antibody titres and DTH responses(Table 2). In particular an adjuvant marketed under trade designationMontanide ISA-50 and available from Seppic, Paris, France has been foundto be suitable. TABLE 2 Animal DTH 24 Hour DTH 48 Hour Antibody LevelsGROUP Number Response Response (450 nm) CON- 19 0 0 0.061 TROL 11 0 00.010 (Un- 1 — — 0.005 vaccin- 15 0 0 0.038 nated) 7 0 0 0.005 QUIL A18 + 0 0.753 25 + 0 0.788 17 0 0 0.638 168 — — 0.642 VEG. 169 +++ 00.316 OIL 22 0 0 0.621 4 + 0 0.666 5 + — 0.239 13 +++ ++ 0.457 MIN. 14+++ ++ 1.085 OIL 5 +++ ++ 1.024 23 +++ + 0.864 15 +++ 0 0.975 21 + —0.954

[0177] Protection Pen Trial

[0178] Groups of 9 young piglets, 6 weeks of age, were immunised withpurified and semi-purified antigens as shown in Table 3 below. Theantigens were purified on reversed-phase HPLC using a formic acidsolvent system with an acetonitrile gradient.

[0179] Antigens were resolubilised in 4 Molar urea before incorporationin mineral oil adjuvant.

[0180] The immunisation schedule is as shown in Table 2. TABLE 3Protocol for Pen Trial of Antigens of Mycoplasma HyopneumoniaeVACCINATIONS & BLEEDS Treatment Day Number 1st Vaccination  0 2ndVaccination 14 3rd Vaccination 50 Infectious Challenge 64 Slaughter 91ANTIGEN DOSES Partly Purified 1st & 2nd Vaccns. 50 μg COMPLEX ANTIGEN/DOSE 52 kD 3rd Vaccn. - 220 μg PARTIALLY PURIFIED ANTIGEN/DOSE(Purified)74 + 52 kD 1st Vaccn. 20 μg total protein/DOSE 2nd Vaccn. 13μg total protein/DOSE 3rd Vaccn. 17 μg total protein/DOSE (Purified) 48KD 1st Vaccn. 20 μg/DOSE 2nd Vaccn. 18 μg/DOSE 3rd Vaccn. 27 μg/DOSE

[0181] ALL PROTEIN ESTIMATIONS DONE BY “BCA” PROTEIN ASSAY (Pierce,Ill., U.S.A.

[0182] Protection from infection with Mycoplasma hyopneumoniae wasassessed by infectious challenge 2 weeks after the final immunisation.Infectious challenge was achieved by intranasal administration of 10 mlof a 10% (w/v) lung homogenate, prepared from infected lung, and byhousing test piglets with previously infected piglets. Four weeks afterinfectious challenge, the animals were killed and the extent and degreeof lung lesions assessed (Table 4). TABLE 4 Pen Trial of Antigens ofMycoplasma Hyponeumoniae No. Pneumonia Medium Lung % Reduction Group No.Free (%) Lesion Score (from Median) Controls 1 (11) 13 0% 52 kD 0 (0)  561% 74 ÷ 52 3 (33) 6.75 48% kD 48 kD 2 (22) 6.25 52%

[0183] Reference

[0184] Warren H. S. and Chedid, L. A., Future Prospects for VaccineAdjuvants CRC Critical Reviews in Immunology 8: 83-108, 1988.

[0185] Finally, it is to be understood that various other modificationsand/or alterations may be made without departing from the spirit of thepresent invention as outlined herein.

1. A putative protective antigen against a Mycoplasma, prepared by amethod including providing a sample of a Mycoplasma; an antibody probeincluding at least one antibody against a Mycoplasma produced by amethod including: providing a biological sample taken a short time afteran immune animal has been challenged with a Mycoplasma or Mycoplasmaextract taken from the infection site or an area of a lesion or an areaclose to the infection site or lesion; isolating cells from thebiological sample; culturing cells in vitro in a suitable culturemedium; and harvesting antibodies produced from said cells; probing theMycoplasma sample with the antibody probe to detect at least oneantigen; and isolating the antigen detected.
 2. A putative protectiveantigen according to claim 1 wherein the Mycoplasma is Mycoplasmahyopneumoniae.
 3. A putative protective antigen against Mycoplasmahyopneumoniae, or related infections, selected from the group ofantigens having approximate molecular weights of 110-114, 90-94, 72-75,60-64, 52-54 and 46-48 kilodaltons (kD), as herein described, mutants,derivatives and fragments thereof.
 4. A putative protective antigenaccording to claim 3 which is a surface protein.
 5. A putativeprotective antigen according to claim 3 or 4 which is a surfacelipo-protein or membrane protein.
 6. A putative protective antigenaccording to any one of claims 3-5 having approximate molecular weightof 110-114, 90-94, 74, 62, 52 and 48 kD.
 7. A putative protectiveantigen according to claim 3 wherein the antigen in the 72-75 kD regioncontains the following N-terminal amino acid sequence:AGXLQKNSLLEEVWYLAL
 8. A putative protective antigen according to claim 7further including one or more of the following N-terminal amino acidsequences: AKNFDFAPSIQGYKKIAHEL NLKPEQILQLLG LLKAEXNKXIEEINTXLDN
 9. Aputative protective antigen according to claim 3 wherein the antigen inthe 60-64 kD region contains the following N-terminal amino acidsequence: MKLAKLLKGFX(N/L)(M/V)IK ADP(F/I)(R/E)Y(V/A)PQG(Q/A)X(M/N)VG10. A putative protective antigen according to claim 3 wherein theantigen in the 52-54 kD region contains the following N-terminal aminoacid sequence: AGXWAKETTKEEKS
 11. A putative protective antigenaccording to claim 10 further including one or more of the followingN-terminal amino sequences: AWVTADGTVN AIVTADGTVNDNKPNQWVRKY
 12. Aputative protective antigen according to claim 3 wherein the antigen inthe 46-48 kD region contains the following N-terminal amino acidsequence: AGXGQTESGSTSDSKPQAETLKHKV
 13. A putative protective antigenaccording to claim 12 further including one or more of the followinginternal amino acid sequences: TIYKPDKVLGKVAVEVLRVLIAKKNKASRAEQAITKLKLEGFDTQ KNSQNKIIDLSPEG
 14. An isolated nucleic acid fragmentencoding a putative protective antigen against Mycoplasma hyopneumoniaeor related infections, said nucleic acid fragment including thefollowing sequence, mutants, derivatives, recombinants and fragmentsthereof: 10                 20         30         40         501234567890 1234567890 1234567800 1234567890 1234567890 ATGAAAAAAATGCCACTATA CCAGAGGAAA GAGCAGTATA TAAAATAATT 50 AAAATTACAT TTTCTTCATTTGCGCCAGAA TTTTTAAGAA TTAGTACATT 100 AAAAAGTAGA ACAAAAGTTA TTAATGTAAACATTAGCGCA ATCCTTAAGA 150 AAAAATTAAA AGTTTTATCT ATTTTTTTTA ATCGAAATACAACCAGGCAT 200 AAATCTTTGT CAGTATTTAT CAAGTCAGTA TTTTTTCATT ATTTCTACTA250 AATATTATAT TGAATTTGCA TTTTACATAA TCTAAAATTT TACATTTTTT 300TATAACAATT TTTAAAAATT ACTATTTAAT TTATAGTATT TTTTTATTTT 350 TTAGTCTAAATTATAAAATT ATCTTGAATT TTATTTGAAT TTTTATAATT 400 TAGTACTAAA AAATACAAATATTTTTTCCT ATTCTAAGAA AAATTCATTT 450 TTTAAAAAAA ATTGATTTTT ATAGTATAATTTGTTTGTAT AATTGAATAA 500 ACTTGATTTG AAAGGGAACA AAATGAAAAA AATGCTTAGAAAAAAATTCT 550 TGTATTCATC AACTATTTAT GCAACTTCGC TTGCATCAAT TATTGCATTT500 GTTGCAGCAG GTTGTGGACA GACAAAATCA GGTTCAACTT CTGATTCTAA 650ACCACAAGCC GAGAAGCTAA AACATAAAGT AAGTAATGAT TCTATTCGAA 700 TAGCACTAACCGATCCGGAT AATCCTCGAT GAATTAGTGC CCAAAAAGAT 750 ATTATTTCTT ATGTTGATAAAACAGAGGCA GCAACTTCAA CAATTACAAA 800 AAACCAGGAT GCACAAAATA ACTAACTCACTCAGCAAGCT AATTTAAGCC 850 CAGCGCCAAA AGAATTTATT ATTACCCCTG AAAATGGAAGTGGAGTTGGA 900 ACTACTGTTA ATACAATTGC TGATAAAGGA ATTCCGATTG TTGCCTATGA950 TCGACTAATT ACTGGATCTG ATAAATATGA TTGGTATGTT TCTTTTGATA 1000ATGAAAAAGT TGGTGAATTA CAAGGTCTTT CACTTGCTGC GGGTCTATTA 1050 GGAAAAGAAGATGGTGCTTT TGATTCAATT GATCAAATGA ATGAATATCT 1100 AAAATCACAT ATGCCCCAAGAGACAATTTC TTTTTATACA ATCGCAGGTT 1130 CCCAAGATGA TAATAATTCC CAATATTATTATAATGGTGC AATGAAAGTA 1200 CTTAAAGAAT TAATGAAAAA TTCGCAAAAT AAAATAATTGATTTATCTCC 1250 TGAAGGCGAA AATGCTATTT ATGTCCCAAG ATGAAATTAT GGAACTGCCG1300 GTCAAAGAAT CCATCTTTTT CTAACAATTA ACAAAGATCC AACAGGTGGT 1350AATAAAATCA AAACTGTTGG TTCAAAATCA GCTTCTATTT TCAAAGGATT 1400 TCTTGCCCCAAATGATGGAA TGGCCGAACA AGCAATCAAC AAATTAAAAC 1450 TTGAAGGGTT TGATACCCAAAAAATCTTTG TAACTCGTCA AGATTATAAT 1500 GATAAAGCCA AAACTTTTAT CAAAGACGGCGATCAAAATA TGACAATTTA 1550 TAAACCTGAT AAAGTTTTAG GAAAAGTTGC TGTTGAAGTTCATCGGGTTT 1600 TAATTGCAAA GAAAAATAAA GCATCTAGAT CAGAAGTCAA AAACGAACTA1650 AAAGCAAAAC TACCAAATAT TTCATTTAAA TATGATAATC AAACATATAA 1700AGTACAAGGT AAAAATATTA ATACAATTTT AGTAAGTCCA CTAATTGTTC 1750 CAAAAGCTAATATTGATAAT CCTGATGCCT AA 1752


15. An isolated nucleic acid fragment according to claim 14 encodingputative protective antigen wherein the antigen is in the 46-48 kDregion including the following nucleic acid sequence, mutants,derivatives, recombinants and fragments thereof:10                 20         30         40         50 12345678901234567890 1234567890 1234567890 1234567890 ATGAAAAAAA TGCCACTATACCAGAGGAAA GAGCAGTATA TAAAATAATT 50 AAAATTACAT TTTCTTCATT TGCGCCAGAATTTTTAAGAA TTAGTACATT 100 AAAAAGTAGA ACAAAAGTTA TTAATGTAAA CATTAGCGCAATCCTTAAGA 150 AAAAATTAAA AGTTTTATCT ATTTTTTTTA ATCGAAATCC AACCAGGCAT200 AAATCTTTGT CAGTATTTAT CAAGTCGGTA TTTTTTCATT ATTTCTACTA 250AAATATTATT TGAATTTGCA TTTTCCATAA TCTAAAATTT TACATTTTTT 300 TATAACAATTTTTAAAAATT ACTCTTTAAT TTATAGTATT TTTTTATTTT 350 TTAGTCTAAA TTATAAAATTATCATGAATT TTATTTGAAT TTTTATAATT 400 TAGTACTAAA AAATACAAAT ATTTTTTCCTATTCTAAGAA AAATTCATTT 450 TTTAAAAAAA ATTGATTTTT ATAGTATAAT TTGTTTGTATAAATTGAATA 500 ACTTGATTTG AAAGGGAACA AAATGAAAAA AATGCTTAGA AAAAAATTCT550 TGTATTCATC AGCTATTTAT GCAACTTCGC TTGCATCAAT TATTGCATTT 600GTTGCAGCAG GTTGTGGACA GACAGAATCA GGTTCAACTT CTGATTCTAA 650 ACCACAAGCCGAGACGCTAA AACATAAAGT AAGTAATGAT TCTATTCGAA 700 TAGCACTAAC CGATCCGGATAATCCTCGAT GAATTAGTGC CCAAAAAGAT 750 ATTATTTCTT ATGTTGAAGA AACAGAGGCAGCAACTTCAA CAATTACAAA 800 AAACCAGGAT GCACAAAATA ACTGACTCAC TCAGCAAGCTAATTTAAGCC 850 CAGCGCCAAA AGGATTTATT ATTGCCCCTG AAAATGGAAG TGAAGTTGGA900 ACTGCTGTTA ATACAATTGC TGATAAAGGA ATTCCGATTG TTGCCTATGA 950TCGACTAATT ACTGGATCTG ATAAATATGA TTGGTATGTT TCTTTTGATA 1000 ATGAAAAAGTTGGTGAATTA CAAGGTCTTT CACTTGCTCC GGGTCTATTA 1050 GGAAAAGAAG ATGGTGCTTTTGATTCAATT GATCAAATGA ATGAATATCT 1100 AAAATCACAT ATGCCCCAAG AGACAATTTCTTTTTATACA ATCGCGGGTT 1150 CCCAAGATGA TAATAATTCC CAATATTTTT ATAATGGTGCAATGAAAGTA 1200 CTTAAAGAAT TAATGAAAAA TTCGCAAAAT AAAATAATTG ATTTATCTCC1250 TGAAGGCGAA AATGCTGTTT ATGTCCCAGG ATGAAATTAT GGAACTGCCG 1300GTCAAAGAAT CCAATCTTTT CTAACAATTA ACAAAGATCC AGCAGGTGGT 1350 AATAAAATCAAAGCTGTTGG TTCAAAACCA GCTTCTATTT TCAAAGGATT 1400 TCTTGCCCCA AATGATGGAATGGACGAACA AGCAATCACC AAATTAAAAC 1450 TTGAAGGATT TAATACCCAA AAAATCTTTGTAACTCGTCA AGATTATAAT 1500 GATAAAGCCA AAACTTTTAT CAAAGACGGC GATCAAAATATGACAATTTA 1550 TAAACCTGAT AAAGTTTTAG GAAAAGTTGC TGTTGAAGTT CTTCGGGTTT1600 TAATTGCAAA GAAAAATAAA GCATCTAGAT CAGAAGTCGA AAACGAACTA 1650AAAGCAAAAC TACCAAATAT TTCATTTAAA TATGATAATC AAACATATAA 1700 AGTACAAGGTAAAAATATTA ATACAATTTT AGTAAGTCCA GTAATTGTTA 1750 CAAAAGCTAA TGTTGATAATCCTGATGCCT AA 1752


16. A method for producing an antibody against a Mycoplasma includingproviding a biological sample taken a short time after an immune animalhas been challenged with a Mycoplasma or Mycoplasma extract taken fromthe infection site or a area of a lesion or an area close to theinfection site or lesion; isolating cells from the biological sample;culturing cells in vitro in a suitable culture medium; and harvestingantibodies produced from said cells.
 17. A method according to claim 16wherein the biological sample is taken at a predetermined time after theanimal has been challenged with a Mycoplasma, preferably 2 to 7 daysafter challenge.
 18. A method according to claim 16 wherein theculturing of cells in vitro further includes addition of helper factorsto the culture, said helper factors selected from the group includingcytokines used alone or in combination, including Interleukin 1, 2, 3,4, 5, 6, 7 and 8, colony stimulating factors, interferons and any otherfactors that may be shown to have an enhancing effect on specific B cellsecretion.
 19. A method according to any one of claims 16-18 furtherincluding a cell activation step including activating the cells isolatedto proliferate and secrete and/or release antibodies said cellactivation step including adding a cell activating agent to the culturemedium, said cell activating agent selected from the group includingmitogens as herein described and helper factors produced by leukocytes,or their synthetic equivalents or combinations thereof.
 20. A methodaccording to any one of claims 16-19 wherein the antibody is in the formof the supernatant harvested from the culture medium.
 21. An antibodyagainst a Mycoplasma prepared according to the method of any one ofclaims 16-20.
 22. A method of identifying a putative protective antigenassociated with a Mycoplasma, preferably Mycoplasma hyopneumoniae, saidmethod including providing a sample of a Mycoplasma; and an antibodyprobe including at least one antibody against a Mycoplasma; probing theMycoplasma sample with the antibody probe to detect at least oneantigen; and isolating the antigen detected.
 23. A method of purifying aputative protective antigen associated with a Mycoplasma, preferablyMycoplasma hyopneumoniae, said method including providing a crudeantigen mixture; and an antibody against a Mycoplasma immobilized on asuitable support; subjecting the crude antigen mixture to affinitychromatography utilizing the immobilized antibody; and isolating thepurified antigen so formed.
 24. A method for preparing a syntheticantigenic polypeptide against Mycoplasma, preferably Mycoplasmahyopneumoniae, which method includes providing a cDNA library or genomiclibrary derived from a sample of Mycoplasma; and an antibody probeincluding an antibody prepared according to claim 16; generatingsynthetic polypeptides from the cDNA library or genomic library, probingthe synthetic polypeptides with the antibody probe, and isolating thesynthetic antigenic polypeptide detected thereby.